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Protein therapeutics have emerged as a major new class of pharmaceuticals. One important shelf-life-limiting factor of biopharmaceuticals is methionine oxidation, and therefore, it is important that analytical methods are able to thoroughly characterize all possible oxidized variants. Here, we present a fast and sensitive method to perform online methionine oxidation site assignment using granulocyte colony-stimulating factor (filgrastim) as a model. The method is based on top-down MS using the all-ion fragmentation mode of the Exactive benchtop mass spectrometer. Conditions that provide information on the intact mass of the protein as well as on fragment ions that allow unambiguous site assignment of methionine oxidation in filgrastim variants as low as 0.12% of total peak area in a chromatographic time scale were identified. Using this method, we performed methionine oxidation site assignment in H2O2-stressed filgrastim and in filgrastim which was stored at intended conditions, respectively. We show that the relative abundance of oxidation species observed in filgrastim stored under intended conditions differs strikingly from the oxidized species observed after H2O2 stress. Additionally, we report an oxidized filgrastim variant that has not been previously described in the literature.

Top-down MS for rapid methionine oxidation site assignment in filgrastim